The goal of this application is to develop new approaches to monitor norepinephrine and serotonin (5-HT) in the brain of behaving rats with subsecond time resolution. The research plan is based upon our prior development of an electrochemistry-based method to monitor dopamine in behaving animals. Norepinephrine is a neuro-transmitter in the aversion related circuitry in the brain. 5-HT plays a central role in the regulation of mood and emotion. Both are electroactive and amenable to detection with fast-scan cyclic voltammetry at carbon-fiber microelectrodes. Three specific aims focus on new technology (1, 3, and 5), to be evaluated in anesthetized rats, and two others apply these tools in freely moving animals (2 and 4). They are: 1. Rapid electroanalysis of norepinephrine in the brain. The cyclic voltammetry of norepinephrine is distinct from all other neurochemicals except dopamine. Pharmacological and neuroanatomical strategies are set forth to distinguish the two catechol-amines. 2. Characterization of norepinephrine neurotransmission in behaving animals. The method in Aim 1 will be adapted for use in freely moving animals using the procedures and protocols previously developed for dopamine. The behavior to be examined is the response to unanticipated aversive conditions. 3. Rapid electroanalysis of 5-HT in the brain. Cyclic voltammograms of 5- HT distinguish it from other brain substances. However, oxidation of the major metabolite of 5- HT, 5-hydroxyindoleacetic acid, forms passivating films on the electrode surface. Cation exchange films will be used to protect the surface along with a judicious selection of the voltammetric waveform. 5-HT release will be monitored in the substantia nigra reticulata during electrical stimulation of serotonergic cell bodies. 4. 5-HT neurotransmission in behaving animals. The voltammetric method for 5-HT developed in Aim 3 will be adapted for use in freely moving animals. 5-HT release and uptake will be characterized along with the in vivo action of specific serotonergic drugs. 5. Iontophoresis coupled with voltammetry. To investigate the factors that modulate neurotransmitter concentrations, reagents need to be delivered reagents directly to the measurement site. We propose to continue the development and use of iontophoresis using small capillaries attached to the shank of a carbon-fiber microelectrode.